Insect resistance using inhibition of gene expression

ABSTRACT

The current invention provides methods to silence insect genes by using unpackaged dsRNA or siRNA, in one embodiment such dsRNA or siRNA is present in plant vascular tissue, preferably phloem, more particularly phloem sap, and the insect is a plant sap-sucking insect. Also provided are DNA sequences which when transcribed yield a double-stranded RNA molecule capable of reducing the expression of an essential gene of a plant sap-sucking insect, methods of using such DNA sequences and plants or plant cells transformed with such DNA sequences. Also provided is the use of cationic oligopeptides that facilitate the entry of dsRNA or siRNA molecules in insect cells, such as plant sap-sucking insect cells.

INTRODUCTION

Several approaches are currently available to obtain plants withincreased resistance to plant insect pests. However, for plantsap-sucking insects such as aphids, planthoppers, stinkbugs, andwhiteflies, only limited options are currently available to protectplants using transgenic approaches. Plants expressing Bacillusthuringiensis toxins targeted to Lepidopteran insects have not beenshown to possess enhanced resistance towards plant sap-sucking insects(Rao et al., 1998).

dsRNA technology has been proven to be highly specific and highlyeffective in silencing endogenous genes in several organisms. However,the dsRNA provided to date is typically packaged in a bacterial or yeastcell or in transfection promoting agents such as liposomes. In thisinvention, it has now been shown that naked, unpackaged dsRNA can beused to silence genes in plant sap-sucking insects.

Plant sap-sucking insects typically feed from the sap in the vascularsystem of plants which they tap into with the stylets of theirproboscis, causing a reduction in plant vitality and the spreading ofseveral plant viral diseases. Plant sap-sucking insects typically have ashort life-cycle and are capable of building up immense populations veryquickly on a host plant.

BACKGROUND OF THE INVENTION

Published PCT application WO 00/01846 generally refers to a method ofalleviating pest infestation of plants, which method comprises a)identifying a DNA sequence from said pest which is critical either forits survival, growth, proliferation or reproduction, b) cloning saidsequence or a fragment thereof in a suitable vector relative to one ormore promoters capable of transcribing said sequence to RNA or dsRNAupon binding of an appropriate transcription factor to said promoters,and c) introducing said vector into the plant. The plant pests referredto in this published PCT application are nematodes.

U.S. Pat. No. 6,326,193 refers to the use of recombinant insect virusessuch as baculoviruses expressing dsRNA to silence selected insect genes.

Published PCT application WO 99/32619 describes generally that dsRNA maybe used to reduce crop destruction by other plant pathogens such asarachnids, insects, nematodes, protozoans, bacteria, or fungi. Thisapplication shows that E. coli bacteria expressing dsRNAs can conferspecific inhibitory effects on C. elegans nematode larvae that feed onthese bacteria.

Timmons et al. (2001) describe that ingestion of bacteria expressingdsRNAs can produce genetic interference in the nematode Caenorhabditiselegans. These authors describe that from a bioengineering perspective,it may be possible to use a bioengineered dsRNA comestible to modifygene expression in any organism which has a gene-specific dsRNA responsesimilar to that of C. elegans. To respond to such an intervention, thattarget organism would need to have both a dsRNA-response mechanism(RNAi) and a dsRNA uptake/spreading mechanism that would produceresponses such as those observed in C. elegans.

Similarly, inhibition of gene expression after ingestion of bacteriaexpressing double-stranded RNA was also reported for freshwaterplanarian flatworms (Newmark et al., 2003).

Published PCT patent application WO 2004/005485 describes the use ofvectors comprising sequences designed to control plant-parasiticnematodes by RNA interference, and transgenic plants transformed withsuch vectors.

Published US patent application 20030180945 generally describes chimericgenes capable of producing antisense or sense RNA equipped with aprokaryotic promoter suitable for expression of the antisense or senseRNA in a particular prokaryotic host. The prokaryotic host can be usedas a source of antisense and/or sense RNA, e.g., by feeding it to ananimal, such as a nematode or an insect, in which the silencing of thetarget gene is envisioned and monitored by reduction of the expressionof the reporter gene. The target gene and reporter genes should be genespresent in the cells of the target eukaryotic organism, and not in theprokaryotic host organism.

Published US patent application 20030154508 describes a method for pestcontrol comprising exposing said pest to a compound which disrupts,within said pest, a cation-amino acid transporter/channel protein.Generally described are plant cells genetically modified to produce atleast one double-stranded RNAi that is designed to be taken up by pestsduring feeding to block expression (or the function) of a target gene.It is described that RNAi can be used to reduce or prevent messagetranslation in any tissue of the pest because of its ability to crosstissue and cellular boundaries. It is further described that RNAi thatis contacted with a pest by soaking, injection, or consumption of a foodsource will cross tissue and cellular boundaries, and that RNAi can alsobe used as an epigenetic factor to prevent the proliferation ofsubsequent generations of pests.

Published PCT patent application WO 01/37654 generally describes dsRNAtargeted against piercing-sucking insects and chewing insects, and thatdouble stranded RNAs intended to confer sap-sucking insect resistancewould preferentially be expressed in plant tissues on which such insectsfeed, e.g., primary and secondary phloem elements, and would be taken upby the insect via its sucking mechanism, e.g., its stylet. The onlyinsect for which application of dsRNA is exemplified is Manduca sexta, aLepidopteran insect. Susceptibility of this insect to RNAi wasdetermined by treating larvae with dsRNA by either feeding or by directinjection. The results from these experiments show that with dsRNAsequences derived from three separate genes, injection into M. sextaleads to substantial decrease in expression of the endogenous gene. Noresults of the feeding experiment are reported.

Published PCT patent application WO 02/14472 describes methods forinhibiting target gene expression, by expressing in a cell a nucleicacid construct comprising an inverted repeat and a sense or antisenseregion having substantial sequence identity to a target gene, whereinthe inverted repeat is unrelated to the target gene. Listed as one ofthe possible targets are insects such as sucking insects.

US published patent application 20030150017 describe the use of RNAmolecules homologous or complementary to a nucleotide sequence of aplant pest such as nematodes and insects. Application of RNAi to a modelinsect species, the lepidopteran insect Helicoverpa armigera, in themodel plant lettuce, is suggested in the Examples, but no feedingexperiment is done and no results are reported.

Published PCT patent application WO 03/004644 A1 describes that theevolutionary distance between nematodes and insects is considerable, andthat there is no reason to assume that while feeding dsRNA to C. eleganswas successful, it would be a technique easily transferable to insects.It further describes the use of dsRNA technology to arthropods, andshows that direct feeding of naked, unpackaged, dsRNA failed to producean RNAi phenotype in Drosophila melanogaster and Helicoverpa armigera,indicating that transfection promoting agents such as liposomes werenecessary for effective transfection in these species. This applicationgenerally envisages that in arthropods with a simple digestive systemnaked dsRNA may be effective in obtaining gene silencing. No results ofthe delivery of naked dsRNA to any arthropod are included in thisapplication.

Gura (2003) describes that while in C. elegans feeding of E. colistrains engineered to produce the double-stranded RNA can trigger RNAi,in the insect Drosophila the feeding of yeast cells engineered to makedouble-stranded RNA failed to work.

Also, Rajagopal et al. (2002) described that in Spodoptera liturainsects, experiments to introduce dsRNA into neonate larvae of S. lituraby soaking them in dsRNA solution or by feeding through diet wereunsuccessful, since no reduction in transcript levels was detected.

Rao et al. (1998) describe that expression of a snowdrop lectin intransgenic rice plants can confer resistance to rice brown planthopper.

Hence, the prior art does not show that naked, unpackaged dsRNA or siRNAcan be used to obtain gene silencing in insects through feeding.

SUMMARY OF THE INVENTION

The current invention provides means to silence insect genes by usingunpackaged dsRNA, in one embodiment such dsRNA is present in vasculartissue, preferably phloem, more particularly phloem sap, and the insectis a plant sap-sucking insect such as an aphid or a whitefly.

The invention described herein is summarized in the following numberedparagraphs:

1. A chimeric gene comprising the following operably linked DNA:

(a) a plant-expressible promoter;

(b) a DNA region which when transcribed yields a double-stranded RNAmolecule capable of reducing the expression of an essential gene of aplant sap-sucking insect, said RNA molecule comprising a first andsecond RNA region wherein:

(i) said first RNA region comprises a nucleotide sequence of at least 19consecutive nucleotides having at least about 94% sequence identity tothe nucleotide sequence of said endogenous gene;

(ii) said second RNA region comprises a nucleotide sequencecomplementary to said at least 19 consecutive nucleotides of said firstRNA region;

(iii) said first and second RNA region are capable of base-pairing toform a double stranded RNA molecule between at least said 19 consecutivenucleotides of said first and second region;

(c) optionally, a 3′ end region comprising transcription termination andpolyadenylation signals functioning in cells of said plant.

2. The chimeric gene of paragraph 1, wherein said essential gene of saidplant sap-sucking insect is selected from the group consisting of thegenes encoding the following: a gut cell protein, a membrane protein, atranscription factor, an ecdyson receptor, a vATPase, an amino acidtransporter, a peptidylglycine alpha-amidating monooxygenase; a cysteinprotease, an aminopeptidase, a dipeptidase, a sucrase/transglucosidase,a translation elongation factor, the eucaryotic translation initiationfactor 1A, a splicing factor, an apoptosis inhibitor, a tubulin protein,an actin protein, an alpha-actinin protein, a histone, a histonedeacetylase, a cell cycle regulatory protein, a cellular respiratoryprotein; a receptor for an insect-specific hormonal signal, a juvenilehormone receptor, an insect peptidic hormone receptor; a proteinregulating ion balance in the cell, a proton-pump, a Na/K pump, anintestinal protease; an enzyme involved in sucrose metabolism, adigestive enzyme, a trypsin-like protease and a cathepsin B-likeprotease.

3. The chimeric gene of paragraphs 1 or 2, wherein said double-strandedRNA silences the gene corresponding to the DNA sequence of any one ofSEQ ID NO: 5 to 8, SEQ ID NO: 11 or SEQ ID NO:12.

4. The chimeric gene of any one of paragraphs 1 to 3, wherein betweensaid first and second RNA region, a spacer region containing a plantintron is present.

5. The chimeric gene of any one of paragraph 1 to 4, wherein saidessential gene has a portion which occurs with the same sequence or withat least 94% sequence identity in homologous genes of several plantsap-sucking insects.

6. The chimeric gene of any one of paragraph 1 to 5, wherein saidpromoter is a constitutive promoter.

7. The chimeric gene of any one of paragraph 1 to 6, wherein saidpromoter is a vascular-specific or a phloem-specific promoter.

8. The chimeric gene of paragraph 7, wherein vascular- orphloem-specific promoter is selected from the group consisting of: therolC or rolA promoter of Agrobacterium rhizogenes, the promoter of theAgrobacterium tumefaciens T-DNA gene 5, the rice sucrose synthase RSs1gene promoter, the Commelina yellow mottle badnavirus promoter, thecoconut foliar decay virus promoter, the rice tungro bacilliform viruspromoter, the promoter of the pea glutamine synthase GS3A gene, theinvCD111 and invCD141 promoters of the potato invertase genes, thepromoter isolated from Arabidopsis shown to have phloem-specificexpression in tobacco by Kertbundit et al (1991), the VAHOX1 promoterregion, the pea cell wall invertase gene promoter, an acid invertasegene promoter from carrot, the promoter of the sulfate transporter geneSultr1;3, the promoter of a plant sucrose synthase gene, the promoter ofa plant sucrose transporter gene.

9. A plant cell, tissue, or a plant or a plant seed comprising thechimeric gene of any one of paragraphs 1 to 8 or the double-stranded RNAmolecule described in any one of paragraphs 1 to 8.

10. A method to silence a gene of a plant sap-sucking insect, comprisingapplying to the feed of said plant sap-sucking insect unpackaged, nakeddsRNA or siRNA which is targeted to an essential plant sap-sucking gene.

11. The method of paragraph 10, wherein said essential gene is any ofthe genes listed in paragraph 2 above.

12. The method of paragraph 10, wherein said application is byexpression of a dsRNA chimeric gene in phloem cells of a plant.

13. A method to silence a gene in a plant sap-sucking insect,comprising: adding naked, unpackaged dsRNA or siRNA to the diet or feedof said plant sap-sucking insect, wherein said dsRNA or siRNA targetssaid gene.

14. A method of controlling plant sap-sucking insects, comprisingexpressing in the phloem of a plant dsRNA that targets an essentialplant sap-sucking insect gene.

15. The method of paragraph 14 wherein said gene is a gene expressed atleast in the intestine or in gut cells.

16. The method of paragraph 14 wherein said plant sap-sucking insect isan aphid or a whitefly.

17. A plant, comprising stably inserted in its genome, the chimeric geneof paragraph 1, so that said chimeric gene is expressed in the phloem orxylem of said plant.

18. A method of identifying gene function in a plant sap-sucking insect,comprising the step of applying naked, unpackaged dsRNA targeting aplant sap-sucking insect gene to the diet of said insect, and evaluatingphenotypic or biochemical changes in said insect.

19. A method of identification of novel targets for insecticidalcompounds, comprising the steps of: a) applying naked, unpackaged dsRNAor siRNA molecules to the feed or diet of a plant-sap sucking insect; b)analyzing which genes when silenced confer lethality to said insect, c)cloning and characterizing said genes thus analyzed; d) identifyingcompounds that disrupt or inactivate said gene or the RNA or proteinencoded thereby; and e) contacting said compounds with said insect orfeed or diet of said insect to confirm the pesticidal nature of saidcompound.

20. Phloem of a plant, comprising siRNA targeted to an aphid essentialgene.

21. Phloem sap of a plant, comprising siRNA targeted to an aphidessential gene.

22. An aphid gene comprising the sequence of any one of SEQ ID NO:5 to8, SEQ ID NO: 11 or SEQ ID NO:12.

23. The method of paragraph 18 or 19, wherein a cationic oligopeptide ismixed in the diet together with the dsRNA.

24. The method of paragraph 23, wherein said oligopeptide is a 12 aminoacids poly-Arginine peptide.

25. The plant cell, tissue, plant or plant seed of paragraph 9 or 17,which also comprises a chimeric gene encoding a cationic oligopeptide.

26. The plant cell, tissue, plant or plant seed of paragraph 25, whereinsaid oligopeptide is a 12 amino acids poly-Arginine peptide.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention, dsRNA or siRNA is fed to a plantsap-sucking insect without being contained in a cell or atransfection-promoting agent. In one embodiment of the invention, thisfeeding is on a plant which expresses the dsRNA or siRNA so that itenters the sap contained in its vascular system. A “transfectionpromoting agent”, as used herein, refers to a lipid-containing materialthat allows or enhances passage of the cell membrane and hence securesuptake by a cell of an extracellular molecule or compound such as adsRNA, particularly liposomes. Such agents are described in publishedPCT patent application WO 03/004644. For the avoidance of doubt, thecationic oligopeptides used in some embodiments of the current inventionare not included in this definition of transfection-promoting agents. AdsRNA or siRNA delivered without transfection promoting agent is alsoreferred to herein as “naked” and/or “unpackaged” dsRNA or siRNA. Acationic oligopeptide, as used herein, does not “package” dsRNA or siRNA(contrary to a transfection promoting agent, such as a liposome) andhence can be supplied together with the dsRNA of siRNA in the case of“unpackaged” delivery of dsRNA or siRNA. The term “chimeric” whenreferring to a gene or DNA sequence is used to refer to a gene or DNAsequence comprising at least two functionally relevant DNA fragments(such as promoter, 5′UTR, coding region, 3′UTR, intron) that are notnaturally associated with each other and/or originate, for example, fromdifferent sources. “Foreign” referring to a gene or DNA sequence withrespect to a plant species is used to indicate that the gene or DNAsequence is not naturally found in that plant, or is not naturally foundin that genetic locus in that plant. The term “foreign DNA” will be usedherein to refer to a DNA sequence as it has incorporated into the genomeof a plant as a result of transformation.

Two sequences or genes (or parts thereof) which are “homologous” or“similar”, as used herein, are similar in sequence to such a degree thatwhen the two sequences are aligned, the percent sequence identity, i.e.,the number of positions with identical nucleotides divided by the numberof nucleotides in the shorter of the sequences, is higher than 70%,higher than 85%, higher than 90%, higher than 95%, or is between 96% and100%. Homologous genes or parts thereof, as used herein, do not requireany evolutionary relationship, though genes related to each other byindependent evolution from the same ancestral gene can be homologousgenes as used herein. As used herein, a gene “homolog” (or homologousgene) can be a gene paralog (or a paralogous gene) and a gene ortholog(or an orthologous gene). In one embodiment of the invention ahomologous gene is an orthologous gene (i.e., a similar gene in adifferent species likely evolved from a common ancestor and whichnormally has retained essentially the same or the same function).Sequences or parts of sequences which have “high sequence identity”, asused herein, refers to the number of positions with identicalnucleotides divided by the number of nucleotides in the shorter of thesequences, being higher than higher than 95%, or between 96% and 100%. Atarget gene, or at least a part thereof, as used herein, preferably hashigh sequence identity to the dsRNA of the invention in order forefficient gene silencing to take place in the target pest. Identity insequence of the dsRNA or siRNA with a part of the target gene RNA isincluded in the current invention but is not necessary.

For the purpose of this invention, the “sequence identity” of tworelated nucleotide or amino acid sequences, expressed as a percentage,refers to the number of positions in the two optimally aligned sequenceswhich have identical residues (×100) divided by the number of positionscompared. A gap, i.e., a position in an alignment where a residue ispresent in one sequence but not in the other is regarded as a positionwith non-identical residues. The alignment of the two sequences isperformed by the Needleman and Wunsch algorithm (Needleman and Wunsch1970). A computer-assisted sequence alignment can be convenientlyperformed using a standard software program such as GAP which is part ofthe Wisconsin Package Version 10.1 (Genetics Computer Group, Madison,Wis., USA) using the default scoring matrix with a gap creation penaltyof 50 and a gap extension penalty of 3.

For the purpose of the invention, the “complement of a nucleotidesequence X” is the nucleotide sequence which would be capable of forminga double stranded DNA molecule with the represented nucleotide sequence,and which can be derived from the represented nucleotide sequence byreplacing the nucleotides by their complementary nucleotide according toChargaff's rules (A< >T; G< >C) and reading in the 5′ to 3′ direction,i.e., in opposite direction of the represented nucleotide sequence.

As used herein, “dsRNA” refers to double-stranded RNA that comprises asense and an antisense portion of a selected target gene (or sequenceswith high sequence identity thereto so that gene silencing can occur),as well as any smaller double-stranded RNAs formed therefrom by RNAse ordicer activity. Such dsRNA can include portions of single-stranded RNA,but contains at least 19 nucleotides double-stranded RNA. In oneembodiment of the invention, the dsRNA is a hairpin RNA which contains aloop or spacer sequence between the sense and antisense sequences of thegene targeted, preferably such hairpin RNA spacer region contains anintron, particularly the rolA gene intron (Pandolfini et al., 2003), thedual orientation introns from pHellsgate 11 or 12 (see WO 02/059294(incorporated by reference herein), and SEQ ID NO: 25 and 15 therein) orthe pdk intron (Flayeria trinervia pyruvate orthophosphate dikinaseintron 2; see WO99/53050 incorporated by reference). “siRNAs” as usedherein are small interfering (double-stranded) RNA molecules of 16-30bp, 19-28 bp, or 21-26 bp, e.g., the RNA forms that can be created byRNAseIII or dicer activity from longer dsRNA. “siRNAs” as used hereininclude any double-stranded RNA of 19 to 26, or 21 to 24 basepairs thatcan interfere with gene expression when present in a cell wherein suchgene is expressed. siRNA can be synthetically made, expressed andsecreted directly from a transformed cell or can be generated from alonger dsRNA by enzymatic activity. These siRNAs can be blunt-ended orcan have overlapping ends.

In one embodiment of the invention, sense and antisense RNAs can beseparately expressed in vitro or in host cells, e.g., in cells of aplant from different chimeric gene constructs using the same or adifferent promoter or from a construct containing two convergentpromoters in opposite orientation. These sense and antisense RNAs whichare formed, e.g., in the same plant cells, can then combine to formdsRNA or siRNA. It is clear that whenever reference is made herein to adsRNA or siRNA chimeric gene or a dsRNA or siRNA molecule, that suchdsRNA or siRNA formed, e.g., in plant cells, from sense and antisenseRNA produced separately is also included. Also synthetically made siRNAor dsRNA annealing RNA strands are included herein when the sense andantisense strands are present together.

A dsRNA or siRNA “targeting” a plant sap-sucking insect gene, as usedherein, refers to a dsRNA or siRNA that is designed to be identical orhave high sequence identity to an endogenous plant sap-sucking insectgene (a target gene), and as such is designed to silence such gene uponapplication to such insect. One dsRNA can target one or severalhomologous genes in one plant sap-sucking insect or one or severalhomologous genes in different plant sap-sucking insects which can feedon the same host plant.

The dsRNA chimeric gene, encoding a dsRNA targeting a plant sap-suckingessential gene, can be stably inserted in a conventional manner into thegenome of a single plant cell, and the so-transformed plant cell can beused in a conventional manner to produce a transformed plant that hasincreased insect resistance. In this regard, a disarmed Ti-plasmid,containing the dsRNA chimeric gene, in Agrobacterium tumefaciens can beused to transform the plant cell, and thereafter, a transformed plantcan be regenerated from the transformed plant cell using the proceduresdescribed in the art, for example, in EP 0 116 718, EP 0 270 822, PCTpublication WO 84/02913 and published European Patent application (“EP”)0 242 246. Preferred Ti-plasmid vectors each contain the dsRNA chimericgene between the border sequences, or at least located to the left ofthe right border sequence, of the T-DNA of the Ti-plasmid. Of course,other types of vectors can be used to transform the plant cell, usingprocedures such as direct gene transfer (as described, for example in EP0 233 247), pollen mediated transformation (as described, for example inEP 0 270 356, PCT publication WO 85/01856, and U.S. Pat. No. 4,684,611),plant RNA virus-mediated transformation (as described, for example in EP0 067 553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation(as described, for example in U.S. Pat. No. 4,536,475), and othermethods such as the methods for transforming certain lines of corn(e.g., U.S. Pat. No. 6,140,553; Fromm et al., 1990; Gordon-Kamm et al.,1990) and rice (Shimamoto et al., 1989; Datta et al., 1990) and themethod for transforming monocots generally (PCT publication WO92/09696). For cotton transformation, especially preferred is the methoddescribed in PCT patent publication WO 00/71733. For soybeantransformation, reference is made to methods known in the art, e.g.,Hinchee et al. (1988) and Christou et al. (1990) or the method of WO00/42207.

The resulting transformed plant can be used in a conventional plantbreeding scheme to produce more transformed plants with the samecharacteristics or to introduce the dsRNA gene in other varieties of thesame or related plant species. Seeds, which are obtained from thetransformed plants, contain the dsRNA gene as a stable genomic insert.Plants comprising a dsRNA or siRNA in accordance with the inventioninclude plants comprising or derived from root stocks of plantscomprising the dsRNA chimeric gene of the invention, e.g., fruit trees.Hence, any non-transgenic grafted plant parts inserted on a transformedplant or plant part are included in the invention since the RNAinterference signal is transported to these grafted parts and any aphidsfeeding on such grafted plant are similarly affected by the dsRNA orsiRNA of the invention.

A DNA encoding a dsRNA is inserted in a plant cell genome so that thisDNA is downstream (i.e., 3′) of, and operably linked to, aplant-expressible promoter which can direct expression in plant cells.This is preferably accomplished by inserting the dsRNA chimeric gene inthe plant cell genome, particularly in the nuclear or plastid (e.g.,chloroplast) genome.

A ‘plant-expressible promoter’ as used herein refers to a promoter thatensures expression of a dsRNA of the invention in a plant cell. Examplesof promoters directing constitutive expression in plants are known inthe art and include: the strong constitutive 35S promoters (the “35Spromoters”) of the cauliflower mosaic virus (CaMV), e.g., of isolates CM1841 (Gardner et al., 1981), CabbB-S (Franck et al., 1980) and CabbB-JI(Hull and Howell, 1987); promoters from the ubiquitin family (e.g., themaize ubiquitin promoter of Christensen et al., 1992, see also Comejo etal., 1993), the gos2 promoter (de Pater et al., 1992), the emu promoter(Last et al., 1990), actin promoters such as the promoter described byAn et al. (1996), the rice actin promoter described by Zhang et al.(1991); promoters of the Cassaya vein mosaic virus (NO 97/48819,Verdaguer et al. (1998)), the pPLEX series of promoters fromSubterranean Clover Stunt Virus (WO 96/06932, particularly the S4 or S7promoter), a alcohol dehydrogenase promoter, e.g., pAdh1S (GenBankaccession numbers X04049, X00581), and the TR1′ promoter and the TR2′promoter (the “TR1′ promoter” and “TR2′ promoter”, respectively) whichdrive the expression of the 1′ and 2′ genes, respectively, of the T-DNA(Velten et al., 1984). Alternatively, a plant-expressible promoter canbe a tissue-specific promoter, i.e., a promoter directing a higher levelof expression in some cells or tissues of the plant, e.g., in greentissues (such as the promoter of the PEP carboxylase). The plant PEPcarboxylase promoter (Pathirana et al., 1997) has been described to be astrong promoter for expression in vascular tissue and is useful in oneembodiment of the current invention. Alternatively, a plant-expressiblepromoter can also be a wound-inducible promoter, such as the promoter ofthe pea cell wall invertase gene (Zhang et al., 1996). A‘wound-inducible’ promoter as used herein means that upon wounding ofthe plant, either mechanically or by insect feeding, typically bypiercing of the plant to access the vascular system in the plantsap-sucking insects, expression of the coding sequence under control ofthe promoter is significantly increased in such plant. It has been shownthat plant sap-sucking insects can also cause plant defense responsessimilar to those observed for other pathogens and wounding (Moran andThompson, 2001). Promoters of certain of such genes induced by plantsap-sucking insects, preferably when their expression is preferentiallyin the vascular tissue, particularly phloem, can also be used inaccordance with the current invention.

In one embodiment of this invention the plant-expressed promoter is avascular-specific promoter such as a phloem-specific promoter. A“vascular-specific” promoter, as used herein, is a promoter which is atleast expressed in vascular cells, or a promoter which is preferentiallyexpressed in vascular cells. Expression of a vascular-specific promoterneed not be exclusively in vascular cells, expression in other celltypes or tissues is possible. A “phloem-specific promoter” as usedherein, is a plant-expressible promoter which is at least expressed inphloem cells, or a promoter which is preferentially expressed in phloemcells. Expression of a phloem-specific promoter need not be exclusivelyin phloem cells, expression in other cell types or tissues, e.g., xylemtissue, is possible. In one embodiment of this invention, aphloem-specific promoter is a plant-expressible promoter at leastexpressed in phloem cells, wherein the expression in non-phloem cells ismore limited (or absent) compared to the expression in phloem cells.Examples of suitable vascular-specific or phloem-specific promoters inaccordance with this invention include but are not limited to thepromoters selected from the group consisting of: the SCSV3, SCSV4,SCSV5, and SCSV7 promoters (Schunmann et al., 2003), the rolC genepromoter of Agrobacterium rhizogenes (Kiyokawa et al., 1994; Pandolfiniet al., 2003; Graham et al., 1997; Guivarc'h et al., 1996, Almon et al;1997), the rolA gene promoter of Agrobacterium rhizogenes (Dehio et al.,1993), the promoter of the Agrobacterium tumefaciens T-DNA gene 5(Korber et al. 1991), the rice sucrose synthase RSs1 gene promoter (Shiet al., 1994), the CoYMV or Commelina yellow mottle badnavirus promoter(Medberry et al., 1992; Zhou et al., 1998), the CFDV or coconut foliardecay virus promoter (Rohde et al., 1994; Hehn and Rhode, 1998), theRTBV or rice tungro bacilliform virus promoter (Yin and Beachy, 1995;Yin et al., 1997), the pea glutamine synthase GS3A gene (Edwards et al.,1990; Brears et al., 1991), the invCD111 and invCD141 promoters of thepotato invertase genes (Hedley et al., 2000), the promoter isolated fromArabidopsis shown to have phloem-specific expression in tobacco byKertbundit et al (1991), the VAHOX1 promoter region (Tomero et al.,1996), the pea cell wall invertase gene promoter (Zhang et al., 1996),the promoter of the endogenous cotton protein related to chitinase of USpublished patent application 20030106097, an acid invertase genepromoter from carrot (Ramloch-Lorenz et al., 1993), the promoter of thesulfate transporter gene Sultr1;3 (Yoshimoto et al., 2003), a promoterof a sucrose synthase gene (Nolte and Koch, 1993), and the promoter of atobacco sucrose transporter gene (Kuhn et al., 1997). Also, any promoterhomologous to (or having a high sequence identity to) any of the abovepromoters and also exhibiting phloem-specific expression, as definedherein, can be used. The selection of a particular promoter can dependon the insect species mainly targeted (e.g., a specific insect targetedcan be mostly a leaf feeder or mostly a root feeder, allowing differentpromoter specificities to be used), and on the expression level andtissue distribution desired. These promoters can be combined withenhancer elements, they can be combined with minimal promoter elements,or can comprise repeated elements to ensure the expression profiledesired.

Elements which can be used to increase expression in plant cells can be:introns at the 5′ end or 3′ end of the chimeric gene, e.g., the hsp70intron, promoter enhancer elements, duplicated or triplicated promoterregions, 5′ leader sequences different from another transgene ordifferent from an endogenous (plant host) gene leader sequence, 3′trailer sequences different from another transgene used in the sameplant or different from an endogenous (plant host) trailer sequence.

The dsRNA gene of the invention can be inserted in the plant genome sothat the inserted gene part is upstream (i.e., 5′) of suitable 3′ endtranscription regulation signals (i.e., transcript formation andpolyadenylation signals). This is preferably accomplished by insertingthe dsRNA chimeric gene in the plant cell genome. Preferredpolyadenylation and transcript formation signals include those of thenopaline synthase gene (Depicker et al., 1982), the octopine synthasegene (Gielen et al., 1984), the SCSV or the Malic enzyme terminators(Schunmann et al., 2003), and the T-DNA gene 7 (Velten and Schell,1985), which act as 3′-untranslated DNA sequences in transformed plantcells.

The dsRNA chimeric gene can optionally be inserted in the plant genomeas a hybrid gene, containing several dsRNA regions which targetdifferent genes in the same or different plant sap-sucking insects, orwhich target different portions of the same gene. Also, it is convenientto include in the transforming DNA of the invention also a selectable orscorable marker gene, such as the bar or the neo gene, so thattransformed plants can easily be selected by application of glufosinateor kanamycin, respectively, as is well known in the art.

Although plant delivery of a dsRNA or siRNA is an embodiment of thisinvention, in accordance with this invention, application of the dsRNAor siRNA of the invention to a plant sap-sucking insect can be done inseveral ways, and need not be by way of a plant expressing a dsRNA orsiRNA. Any method of delivery of siRNA or dsRNA not contained in a cellor a cell-transfection agent (such as a liposome) is included herein,e.g., in vitro produced siRNA or dsRNA applied to an insect diet orfeed.

“Insecticidal activity” of a dsRNA or siRNA, as used herein, refers tothe capacity to obtain mortality in insects when such RNA is fed toinsects, preferably by expression in a recombinant host such as a plant,which mortality is significantly higher than the controls (using anon-insect dsRNA or buffer). “Insect-control” of a dsRNA or siRNA, asused herein, refers to an amount of RNA which is sufficient to limitdamage on a plant by insects feeding on such plant, e.g., by killing theinsects or by inhibiting the insect development, fertility or growth insuch a manner that they provide less damage to a plant, produce feweroffspring, are less fit or more susceptible to predator attack, or thatinsects are even deterred from feeding on such plant.

Information on how to design optimal dsRNA or siRNA sequences once atarget gene is known can be found with commercial providers, e.g., thecompanies Ambion and Cenix BioScience (Ambion Inc., 2130 WoodwardStreet, Austin, Tex. 78744-1832, USA; and see www.ambion.com; and CenixBioScience GmbH, Pfotenhauerstr. 108, 01307 Dresden, Germany, seewww.cenix-bioscience.com). Preferably, the dsRNAs or siRNAs to be usedin this invention target at least one essential plant sap-sucking insectgene, or an essential plant sap-sucking insect gene occurring withoutsignificant sequence divergence (at least in a certain region) in arange of plant sap-sucking insect pests of the host plant concerned. Inone embodiment of this invention, such dsRNAs or siRNAs do not silencegenes of the plant host or of other non-target animals, such as plantsap-sucking insect predators (e.g., ladybird larvae, anthocorid bugs,lacewing, parasitic wasps or hoverfly larvae) or animals such asreptiles, amphibians, birds, or mammals. This can be analyzed inavailable databases, e.g., by a BLAST search (see alsowww.ncbi.nim.nih.gov/BLAST) or by hybridization with existing DNAlibraries of representative non-target organisms. In this respect, whenusing the A. gossypii eIF1A sequence of SEQ ID NO: 5 to transform aplant with a dsRNA chimeric gene, preferably only that portion fromnucleotide position 72 to the end in SEQ ID NO:5 should be used as genetarget in designing the dsRNA molecule, or at least the portion fromnucleotide position 50 to 73 in SEQ ID NO:5 should be avoided in thedsRNA. In one embodiment, a portion of a target sequence is selectedwhich is present in several plant sap-sucking insects of a plant host inidentical sequence or with high sequence identity, e.g., a part of aclass of gene(s) with high sequence homology in several plantsap-sucking insect pests.

A plant sap-sucking insect “target gene”, “an essential gene”, or “agene essential for a plant sap-sucking insect”, as used herein, is agene the silencing of which brings about a decreased growth,development, reproduction or survival of a plant sap-sucking insect. Inone embodiment, the partial or complete silencing of an essential geneof a plant sap-sucking insect results in significant insect mortality orsignificant insect control when such gene is silenced by dsRNA or siRNAcompared to control insects fed on dsRNA or siRNA targeting anon-essential gene or a gene not expressed in the plant sap-suckinginsect. In one embodiment of this invention, the dsRNA or siRNA of theinvention corresponds to an exon in the target gene.

In one embodiment of the invention genes expressed in cells of the plantsap-sucking insect gut tissue or in the midgut are targeted, preferablygenes involved in gut cell metabolism, growth or differentiation. Thesegenes can encode plant sap-sucking insect gut membrane proteins such astransporter molecules or ion pumps, e.g., a vATPase (e.g., a homolog tothe Drosophila gene of Genbank accession number AF143200) or an aminoacid transporter gene. Useful plant sap-sucking insect target genes inaccordance with the invention also include genes encoding the following:a transcription factor; a peptidylglycine alpha-amidating monooxygenase(e.g., a homolog of the Drosophila phm gene described in Jiang et al.,2000); a cystein protease (Cristofoletti et al., 2003), anaminopeptidase (e.g., a gene homologous to a aminopeptidase N geneexpressed in the gut of Drosophila, or a gene encoding the pea aphidaminopeptidase N (Rahbé et al., 1995; Cristofoletti et al., 2003), adipeptidase, a sucrase/transglucosidase (see, Ashford et al. 2000;Cristofoletti et al. (2003)); a translation initiation factor (such asthe eucaryotic translation initiation factor 1A (eIF1A) (e.g., based onthe homolog in Drosophila (Myrick and Dearolf, 2000, Genbank accessionnumber AF169359)), a translation elongation factor (such as a plantsap-sucking insect gene homologous to the Drosophila elongation factor 1alpha gene (Hovemann et al., 1988, Genbank accession number X06869)); asplicing factor (such as a gene homolog of the Drosophila SF1 gene(Marzoui et al., 1999), or Genbank accession number NM_(—)079915); anIAP inhibiting apoptosis (e.g., a homolog of the IAP gene described inHay, 2000 or Genbank accession number AA801628); an (alpha) tubulin(e.g., the homolog of the Drosophila alpha-tubulin gene decribed inMatthews and Kaufman, 1987 or in Genbank accession number All 24284), anactin or alpha-actinin (e.g., the homolog to the Drosophila genedescribed in Fyrberg et al., 1998; Dubreuil and Wang, 2000, and inGenbank accession number NM_(—)058137); a histone protein (e.g., ahomolog of the H2A.F/Z family of Drosophila histone genes (Clarkson andSaint, 1999), such as H2AvD (van Daal and Elgin, 1992, Clarkson et al.,1999; Genbank accession number NM_(—)079795); a histone deacetylase(such as a homolog of the histone deacetylases HDAC1 (Mottus et al.,2000); HDAC3 (Johnson et al, 1998) or HDAC4a (Zeremsky et al., 2003),Genbank accession number AF538713); a cell cycle protein; a proteinessential for cellular respiration; a receptor for an insect-specifichormonal signal, a juvenile hormone receptor (e.g., a plant sap-suckingjuvenile hormone receptor gene described in published PCT patentapplications WO99/36520 and WO01/02436), an insect peptidic hormonereceptor; plant sap-sucking insect homologous genes of the dmHelicase,dmPITP or dmSPL genes of Drosophila identified in WO01/42479; a (partof) ecdysone receptor (e.g., a plant sap-sucking ecdysone receptor geneas the M. persicae or B. tabaci genes described in published PCT patentapplications WO99/36520 and WO01/02436; or an insect gene homologous tothe Drosophila ecdyson receptor gene (see, e.g., the ecdyson receptorgene forms described in Bender et al. (1997); Lam and Thummel, 2000), orto the Drosophila ultraspiracle gene (Henrich et al., 2000)); a proteinessential for regulating ion balance in the cells (e.g., a proton-pump,a NA/K pump, etc.); an intestinal protease; etc. Possible target genesare also other genes encoding enzymes involved in sucrose metabolism inthe plant sap-sucking insect, preferably in the gut, or genes encodingdigestive enzymes such as the trypsin-like protease and the cathepsinB-like protease that were recently described in a Homopteran plantsap-sucking insect, the rice brown planthopper (Foissac et al., 2002),and homologous genes found in other plant sap-sucking insects.

Preferred target sequences in accordance with this invention are theplant sap-sucking insect genes identified using the primers of any oneof SEQ ID NO:1-4 and SEQ ID NO:9 and 10, or plant sap-sucking insectgenes corresponding to or comprising any one of the sequences of SEQ IDNO:5 to 8, SEQ ID NO:11 or SEQ ID NO:12, as well as aphid geneshomologous to or having high sequence identity with the sequences of SEQID NO:5 to 8, SEQ ID NO:11 or SEQ ID NO:12, this includes other portionsof the same aphid genes or genes of other plant sap-sucking insectshaving high sequence identity or homology to the sequences of SEQ IDNO:5 to 8, SEQ ID NO:11 or SEQ ID NO:12. It is preferred that onlyportions of the target gene which are known, i.e., those portions of SEQID NO:5 to 8, SEQ ID NO:11 or SEQ ID NO:12 that have no “n” positions,are used in the design of the chimeric genes of the invention.

In one embodiment of this invention, target genes are plant sap-suckinginsect genes homologous to a gene which when partially or completelysilenced (or otherwise prevented to express a functional protein or RNA)in an insect, e.g., Drosophila, results in a mutant with a lethalphenotype (see, e.g., www.fruitfly.org/p_disrupt/; Spradling et al.,1999; and Adams and Sekelsky, 2002 and references cited therein),particularly when such gene is expressed in insect gut cells,particularly in the gut cells lining the gut lumen, especially gut cellslining the midgut. Homologs of Drosophila genes are easily found byexisting techniques, e.g., PCR amplification of the plant sap-suckinginsect homologous gene using primers targeting a Drosophila essentialgene (e.g., in cDNA or genomic libraries of a preferred target insect),or by routine similarity searches in available DNA sequence databases ofplant sap-sucking insects.

Target genes can also be found using similar sequences of essentialinsect genes isolated and characterized in other non-Drosophila insects.In one embodiment of the invention, the target gene preferably producesa stable mRNA in the aphid. Also, in one embodiment of the invention,although the target gene has to be transcribed in the target insect, forobtaining an optimal silencing effect, it is preferred that the targetgene does not produce abundant amounts of RNA.

To test performance of a certain dsRNA or siRNA in plants, the system asdescribed in published PCT application WO 03/052108 can be used, whereinplants produce dsRNA/siRNA and effects on the aphids growing on suchplants can be assessed. As a control, a different non-essential genethat is absent in aphids, such as a gene encoding a gfp (greenfluorescent protein)-specific dsRNA, is tested in parallel in the sameviral construct.

Also, in the dsRNA chimeric gene of the invention a nuclear localizationsignal can be added as described in published US patent application20030180945 (incorporated herein by reference).

As used herein, nucleotide sequences of RNA molecules may be identifiedby reference to DNA nucleotide sequences of the sequence listing.However, the person skilled in the art will understand whether RNA orDNA is meant depending on the context. Furthermore, the nucleotidesequence is identical except that the T-base is replaced by uracil (U)in RNA molecules.

The length of the first (e.g., sense) and second (e.g., antisense)nucleotide sequences of the dsRNA molecules of the invention may varyfrom about 10 nucleotides (nt) up to a length equaling the length innucleotides of the transcript of the target gene. The length of thefirst or second nucleotide sequence of the dsRNA of the invention can beat least 15 nt, or at least about 20 nt, or at least about 50 nt, or atleast about 100 nt, or at least about 150 nt, or at least about 200 nt,or at least about 500 nt, or at least about 1600 nt. If not allnucleotides in a target gene sequence are known, it is preferred to usesuch portion for which the sequence is known and which meets otherbeneficial requirements of the invention.

It will be appreciated that the longer the total length of the firstnucleotide sequence in the dsRNA of the invention is, the less stringentthe requirements for sequence identity between the total sensenucleotide sequence and the corresponding sequence in the target genebecomes. The total first nucleotide sequence can have a sequenceidentity of at least about 75% with the corresponding target sequence,but higher sequence identity can also be used such as at least about80%, at least about 85%, at least about 90%, at least about 95%, about100%. The first nucleotide sequence can also be identical to thecorresponding part of the target gene. However, it is advised that thefirst nucleotide sequence includes a sequence of 19 or 20, or about 19or about 20 consecutive nucleotides, or even of about 50 consecutivenucleotides, or about consecutive 100 nucleotides, or about 150consecutive nucleotides with only one mismatch, preferably with 100%sequence identity, to the corresponding part of the target gene. Forcalculating the sequence identity and designing the corresponding firstnucleotide sequence, the number of gaps should be minimized,particularly for the shorter sense sequences.

The length of the second (e.g., antisense) nucleotide sequence in thedsRNA of the invention is largely determined by the length of the first(e.g., sense) nucleotide sequence, and may correspond to the length ofthe latter sequence. However, it is possible to use an antisensesequence which differs in length by about 10% without any difficulties.Similarly, the nucleotide sequence of the antisense region is largelydetermined by the nucleotide sequence of the sense region, and may beidentical to the complement of the nucleotide sequence of the senseregion. Particularly with longer antisense regions, it is howeverpossible to use antisense sequences with lower sequence identity to thecomplement of the sense nucleotide sequence, such as at least about 75%sequence identity, or least about 80%, or at least about 85%, moreparticularly with at least about 90% sequence identity, or at leastabout 95% sequence to the complement of the sense nucleotide sequence.Nevertheless, it is advised that the antisense nucleotide sequencealways includes a sequence of 19 or 20, about 19 or about 20 consecutivenucleotides, although longer stretches of consecutive nucleotides suchas about 50 nucleotide, or about 100 nucleotides, or about 150nucleotides with no more than one mismatch, preferably with 100%sequence identity, to the complement of a corresponding part of thesense nucleotide sequence can also be used. Again, the number of gapsshould be minimized, particularly for the shorter (19 to 50 nucleotides)antisense sequences.

In one embodiment of the invention, the DNA molecules according to theinvention may comprise a DNA region encoding a spacer between the DNAregion encoding the first and second nucleotide sequences. As indicatedin WO 99/53050 the spacer may contain an intron to enhance genesilencing. A particularly preferred intron functional in cells of plantsis the pdk intron (Flayeria trinervia pyruvate orthophosphate dikinaseintron 2; see WO99/53050 incorporated by reference), the delta 12desaturase intron from Arabidopsis (Smith et al., 2000) or the intron ofthe rolA gene (Magrelli et al., 1994; Spena and Langenkemper, 1997).

In one embodiment of the invention, the dsRNA molecule may furthercomprise one or more regions having at least 94% sequence identity toregions of at least 19 consecutive nucleotides from the sense nucleotidesequence of the target gene, different from the at least 19 consecutivenucleotides as defined in the first region, and one or more regionshaving at least 94% sequence identity to at least 19 consecutivenucleotides from the complement of the sense nucleotide sequence of thetarget gene, different from the at least 19 consecutive nucleotides asdefined in the second region, wherein these additional regions canbasepair amongst themselves.

Plants to which the current invention can be applied include but are notlimited to the following plants: corn, cotton, rice, soybean, Brassicaspecies plants, Brassica napus, cauliflower, carrot, pea, wheat, barley,rye, tomato, potato, sugarbeet, cut flowers, roses, fruit plants (apple,pear, peach, strawberry, etc.), trees (such as poplar and willow), andlettuce. In one embodiment of the invention, the plants to which theinvention is applied are cotton or rice plants. When cotton is the hostplant of the invention the dsRNA preferably targets genes in one or allof the following sucking insects: Aphis gossypii, Myzus persicae, Lygusbugs, whitefly, stink bugs, thrips, and Creontiades dilutus,particularly Aphis gossypii, Myzus persicae and Creontiades dilutus.

Together with the strategy of this invention, it is preferred to useother tactics for aphid control, such as expression of a snowdrop(Galanthus nivalis) lectin as described by Down et al. (1996), Stoger etal. (1999) and Rao et al. (1998) or the mannose-binding lectins of Royet al. (2002) in the plants of the invention, preferably in the phloemof the plants of the invention, and/or the expression of a proteaseinhibitor such as the soybean Kunitz trypsin inhibitor (Foissac et al.,2002), or the variant mustard trypsin inhibitor Chy8 (Ceci et al., 2003)and similar protease inhibitors active against plant sap-suckinginsects, particularly aphids. Also the timely application of effectivechemical or biological insecticides, and the use of natural aphidpredators are possible tactics to use together with the plants of theinvention. Also existing endogenous resistance genes such as the tomatoMi nematode-resistance gene (which also confers resistance to certainaphid species, see Rossi et al., 1998) or the VAT gene (WO 2004072109)can be used to protect a plant of the invention against aphids andprovide different mechanisms of resistance, hence minimizing chances ofinsect resistance development.

Also, in one embodiment of this invention, the plant co-expressesantibacterial proteins or peptides, preferably in the phloem, to kill ornegatively affect symbiontic bacteria occurring in aphids that arebelieved to provide the aphids with certain essential compounds (such asamino acids) which they may not get sufficiently from feeding on plantsap. These proteins can be any one of the antibacterial peptides orproteins known in the art which are effective in inhibiting bacterialgrowth, and which are preferably provided with a signal forextracellular or for phloem targeting. Such a protein signal sequencecan be found in many proteins which are targeted to the phloem, e.g.,the GNA snowdrop lectin described above. Also, a dsRNA can be expressedin the plants of the invention, which dsRNA targets an essential gene ofsuch symbiontic bacteria, e.g., the essential genes described byShigenobu et al. (2000) for the genome of the bacterial symbiont(Buchnera sp.) of the pea aphid, and homologous forms for symbionts inother aphids.

In one embodiment of this invention, together with the dsRNA or siRNAsof the current invention, a chimeric gene encoding a cationicoligopeptide is expressed in the plants of the invention. With a“cationic oligopeptide” as used herein, is meant an oligopeptide oflarger then 5 and smaller then 40 amino acids with a net positivecharge, and the ability to facilitate transport of siRNA or dsRNA acrossan insect cell membrane. Such cationic oligopeptides are preferablybetween 5 and 40 amino acids long, between 10 and 30 amino acids long,particularly of about 12-18 amino acids long, more particularly 12-18amino acids long, and can bind to the dsRNA of the invention and besidesstabilizing the dsRNA, they facilitate the entry of the dsRNA into thesap-sucking insect cells, particularly their gut cells, preferably theirmidgut cells. In this embodiment, such a cationic oligopeptide, asequence of multiple such cationic peptides separated by spacer aminoacids which are cleavable inside or outside the cell, or a cationicoligopeptide fused to a phloem-targeting signal or to another protein(such as the prosystemin fusion described by Tortiglione et al. (2003))is expressed in the same cells as the dsRNA or siRNA of the invention,preferably but not necessarily using the same promoter (in differentchimeric genes), leading to accumulation of the peptide and the dsRNA ofthe invention in the plant cells and/or phloem. In one embodiment ofthis invention, the cationic oligopeptide is a poly-Arginine 12-mer(Unnamalai et al., 2004). Other cationic oligopeptides that can beco-expressed in any plant include from 5 to 40, 10 to 30, or 12-18 aminoacids long poly-Arginine, poly-Lysine or poly-Histidine peptides ormixtures of any of these 3 basic amino acids, or the penetratin peptide,transportan peptide, TAT peptide, MAP peptide, R7 peptide, pVEC peptide,MPG-delta-NLS peptide, KALA peptide or Buforin 2 peptide, as describedin Jarver and Langel (2004, this publication and the cited referencepapers per peptide are incorporated herein by reference), or otheroligopeptides smaller then 40 amino acids with a net positive charge,and the ability to facilitate transport of siRNA or dsRNA across aninsect cell membrane, preferably the MPG-delta-NLS peptide, the 12-aminoacids poly-Arginine peptide or the TAT peptide. In one embodiment ofthis invention, the chimeric gene encoding the dsRNA and the chimericgene encoding the cationic oligopeptide of the invention are assembledin one transforming DNA, e.g., in one T-DNA insert in an Agrobacteriumplasmid, to secure expression from one locus in the plant. In oneembodiment of this invention, these cationic oligopeptides are usefulfor facilitating transfer of any dsRNA or siRNA into the cells of anyinsect species, not just the sap-sucking insects. Such insects includeinsects used as model species, or insects pests of corn, cotton, rice,soybean, Brassica species plants, oilseed rape, cabbage, cauliflower,carrot, pea, wheat, barley, rye, tomato, potato, sugarbeet, cut flowers,roses, fruit plants (apple, pear, peach, strawberry, etc.), trees (suchas poplar and willow), and lettuce, particularly the european cornborer, cotton bollworms, and corn rootworms, besides the plantsap-sucking insect pests described herein. Particularly such insects areselected from the list consisting of: Drosophila melanogaster, Anophelesspp. insects, Helicoverpa zea, Helicoverpa annigera, Helicoverpapunctigera, Heliothis virescens, Ostrinia nubilalis, Spodopterafrugiperda, Agrotis ipsilon, Pectinophora gossypiella, Scirphophagaincertulas, Cnaphalocrocis medinalis, Sesamia inferens, Chilo partellus,Anticarsia gemmatalis, Plathypena scabra, Pseudoplusia includens,Spodoptera exigua, Spodoptera omithogalli, Epinotia aporema, Rachiplusianu, Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens,Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens,Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis,Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilopolychrysus, Rupela albinella, Diatraea saccharalis, Spodopterafrugiperda, Mythimna unipuncta, Chilo zacconius and Pamara guffata, mostparticularly Chilo suppressalis, Scirpophaga incertulas, Marasmiapatnalis, Cnaphalocrocis medinalis, Agelastica alni, Hypera postica,Hypera brunneipennis, Haltica tombacina, Anthonomus grandis, Tenebriomoliftor, Triboleum castaneum, Dicladispa armigera, Trichispa serica,Oulema oryzae, Colaspis brunnea, Lissorhorptrus oryzophilus, Phyllotretacruciferae, Phyllotreta striolata, Psylliodes punctulata, Entomoscelisamericana, Meligethes aeneus, Ceutorynchus sp., Psylliodeschrysocephala, Phyllotreta undulata, Leptinotarsa decemlineata,Diabrotica undecimpunctata undecimpunctata, Diabrotica undecimpunctatahowardi, Diabrotica barber, and Diabrotica virgifera. Such cationicoligopeptides can also be delivered together with any dsRNA or siRNAmolecules to insect cells in vitro or in cell culture (e.g., by addingthem to the culture medium), or they may be mixed with dsRNA or siRNAmolecules in the insect feed to be fed to insects, with the aim tosilence certain insect target genes. Hence, these peptides are a usefulresearch tool for the analysis of gene function, by mixing them(preferably in 1:1 ratio) with dsRNA or siRNA molecules targetingcertain insect genes, e.g., in a process of pesticide target developmentas described above.

In this embodiment, the dsRNA or siRNA then targets an essential gene ofthis insect, preferably an essential gene that is expressed in the gut,and this dsRNA or siRNA and the cationic oligopeptide of the inventioncan be administered to an insect species cell culture in vitro, to alive insect, preferably to a larval stage thereof, by spraying in afield or by co-expression in a transgenic plant on which such insectspecies tends to feed.

In one embodiment of this invention, the current strategy is used in aplant which has already acquired partial or enhanced resistance to plantsap-sucking insects such as aphids or whiteflies, which plant isobtained by normal breeding and selection, or in a hybrid plant havingbetter overall growth and vigour.

The dsRNAs or siRNAs of the current invention, and the method ofapplying them to the feed of insects such as plant sap-sucking insects,also have applications in insecticide development. New insecticides canbe developed using the process of identifying and validating biologicaltargets against which potential ligands can be screened (e.g., Margolisand Duyk, 1998). Production of new insecticides can be done usingtarget-based discovery approaches. Genes that, when partially orentirely inactivated, kill the organism or significantly affect normaldevelopment when knocked out or repressed represent interesting targets.To identify compounds that have the same effect on the organism,high-throughput screening assays can be established to test compoundsfor their ability to interfere in vitro with the normal activity of thetarget. As such, the invention provides a method of identification ofnovel targets for insecticidal compounds, comprising the steps of: a)applying naked, unpackaged dsRNA or siRNA molecules to the feed or dietof a plant-sap sucking insect; b) analyzing which genes when silencedconfer lethality to said insect, c) cloning and characterizing saidgenes thus analyzed; and optionally: d) identifying compounds thatdisrupt or inactivate said gene or the protein encoded thereby; and e)contacting said chemicals with said insect or feed or diet of saidinsect to confirm the pesticidal nature of said compound. In oneembodiment of this invention, this unpackaged dsRNA or siRNA can besupplied together with a cationic oligopeptide as described herein forapplications to any insect species.

Preferred target pests are plant sap-sucking insects, particularly ofthe order Hemiptera, preferably insects of the suborder Homoptera orHeteroptera, preferably aphids and whiteflies, particularly insects ofthe family of Aphididae. The invention is similarly applicable to anyplant sap-sucking insects feeding from xylem by using a constitutivepromoter or a xylem-specific promoter. Examples of xylem-specificpromoters (i.e., promoter preferentially but not necessarily exclusivelyactive in xylem) include but are not limited to: the Pal2 promoter, e.g.from bean (Leyva et al., 1992), the poxN or poxA promoters from rice(Ito et al., 2000), the PtCesA promoter (Wu et al., 2000), the XCP1promoter (Funk et al., 2002), the cotton curl leaf virus promoter(Yingqiu et al., 2000), the vs-1 promoter element (Torres-Schumann etal., 1996).

“Plant sap-sucking insects” as used herein are insects feeding on plantsusing their sharp mouth parts which can be inserted into a plant to takefluid from the plant vascular system, in one embodiment these areinsects feeding directly on the fluids in the plant vascular system,preferably insects only feeding on the fluids in the plant vascularsystem. In the insertion step, plant cells can also be damaged which mayor may not be used as a food source by the plant sap-sucking insect.These insects are plant pests because their feeding reduces the vitalityof the crops they feed on and they can transmit viral diseases. Also,many such sap-sucking insects secrete a sugar-rich fluid named honeydewthat accumulates on lower plant parts, and such parts soon becomecovered by certain black or brown fungi known as sooty molds, henceinterfering with plant photosynthesis.

Included in such plant sap-sucking insects are aphids or Homopteraninsects of the Aphididae, and plant sap-sucking insects as used hereininclude but are not limited to the peach-potato aphid Myzus persicae,the bean aphid Aphis fabae, the pea aphid Acyrthosiphum pisum, thecabbage aphid Brevicoryne brassicae, the grain aphid Sitobion avenae,the rose-grain aphid Metopolophium dirhodum, the Russian wheat aphidDiuraphis noxia (Mordvilko), the English grain aphid Macrosiphum avenae,the greenbug aphid Schizaphis graminum (Rondani), the carrot aphidCavariella aegopodii, the potato aphid Macrosiphum euphorbiae, thegroundnut aphid Aphis craccivora, the cotton aphid Aphis gossypii, theblack citrus aphid Toxoptera aurantii, the brown citrus apid Toxopteraciidius, the willow aphid Cavariella spp., the corn leaf aphidRhopalosiphum maidis, the aphid Rhopalosiphum padi, the willow leafaphids Chaitophorus spp., the black pine aphids Cinara spp., theSycamore Aphid Drepanosiphum platanoides, the Spruce aphids Elatobiumspp., Aphis citricola, Lipaphis pserudobrassicae (turnip aphid),Nippolachnus piri, the foxglove aphid Aulacorthum solani, the asparagysaphid Brachycorynella asparagi, the brown ambrosia aphid Uroleuconambrosiae, the buckthom aphid Aphis nasturtii, the corn root aphid Aphismaidiradicis, the cresentmarked lily aphid Neomyzus circumflexes, thegoldenglow aphid Dactynotus rudbeckiae, the honeysuckle and parsnipaphid Hyadaphis foeniculi, the leafcurl plum aphid Brachycaudushelichrysi, the lettuce root aphid Pemphigus bursarius, the mint aphidOvatus crataegarius, the artichoke aphid Capitophorus elaeagni, theonion aphid Neotoxoptera formosana, the pea aphid Macrosiphum pisi, therusty plum aphid Hysteroneura setariae, the shallot aphid Myzusascalonicus, the solanum root aphid Smynthurodes betae, the sugarbeetroot aphid Pemphigus betae, the tulip bulb aphid Dysaphis tulipae, thewestern aster root aphid Aphis armoraciae, the white aster root aphidProciphilus erigeronensis.

Also included herein as plant sap-sucking insects are whiteflies orAleyrodidae insects such as Trialeurodes vaporariorum (greenhousewhitefly), the banded wing whitefly Trialeurodes abutilonea, Bemisiatabaci (sweetpotato whitefly) and Bemisia argentifolli (silverleafwhitefly). Also included herein as plant sap-sucking insects arefleahoppers such as Pseudatomoscelis seriatus or cotton fleahopper, andHalticus bractatus or garden fleahopper, Pentatomidae (stink bugs, e.g.,Thyanta spp.), mealybugs (Hemiptera, Coccoidea, Pseudococcidae, e.g.,the citrus mealy bug (genus Pseudococcus)), as well as Delphacidae (orplanthoppers) such as Laodelphax striatellus (small brown planthopper),Nilaparvata lugens (rice brown plant hopper) and Sogatella furcifera(white-backed rice planthopper), and Deltocephalidae (or leafhoppers)such as Flexamia DeLong spp., Nephotettix cincticeps and Nephotettixvirescens, Amrasca bigutulla, and the potato leafhopper Empoascafilament. Also included are scales (also named scale insects) such asAonidiella aurantii (California red scale), Comstockaspis perniciosa(San Jose scale), Unaspis citri (citrus snow scale), Pseudaulacaspispentagona (white peach scale), Saissetia oleae (brown olive scale),Lepidosaphes beckii (purple scale), Ceroplastes rubens (red wax scale)and Icerya purchasi (cottonycushion scale), besides Tingidae (or lacebugs) and Psyllidae insects, and spittle bugs.

Further included as plant sap-sucking insects are Heteropteran insectsand Hemipteran insects of the Auchenorrhyncha that feed from the plants'vascular system, such as sap-sucking insects of the Cicadoidea (such asCicadas), Cercopoidea (spittlebugs or froghoppers), Membracoidea(leafhoppers and treehoppers), and Fulgoroidea (planthoppers), e.g., thecotton seed sucker bug Dysdercus peruvianus (Heteroptera,Pyrrhocoridae), the apple dimpling bug, Campylomma liebknechti(Hemiptera:Miridae) and the green mirid, Creontiades dilutus which arecotton sucking insect pests, and the Lygus bugs (Hemiptera:Miridae,e.g., Lygus hesperus).

In one embodiment of this invention, phloem-feeding plant sap-suckinginsects are targeted with the plants, compositions and methods of theinvention, particularly aphids, planthoppers and whiteflies. However,the invention can be applied to any plant sap-sucking pest or any pestwhich ingests plant sap when feeding on a plant, whether an insect ornot, since the mechanism of dsRNA feeding presented herein can similarlyaffect such plant pest when it ingests dsRNA or siRNA from the plant,e.g., Thrips insects (Thysanoptera, e.g., Thrips tabaci andFrankliniella schultzei). The invention is similarly applicable to othersmall sucking plant pests, e.g., mites and spider mites (e.g.,Tetranychus spp., especially Tetranychus urticae, T. ludeni, T.turkestani, T. pacificus, Tcinnabarinus and T. lambi) sucking onindividual plant cells, since they will also ingest and be affected bythe dsRNA or siRNA produced in the plant of the invention.

This inventions is similarly applicable to any arthropod pest, insect orotherwise, that feeds on plant cells or tissues and that ingests thedsRNA or siRNA produced in the plants of the invention in a naked,unpackaged form, particularly pests of the Homoptera, Hemiptera andArachnida pests.

In one embodiment of the current invention, the dsRNA or siRNA chimericgene construct is present in a plant already expressing an insecticidalprotein, e.g., an insecticidal protein derived from Bacillusthuringiensis. Preferred plants expressing such proteins include but arenot limited to: corn plants containing the MON863 transformation event,corn plants containing the MON810 transformation event, corn plantscontaining the Bt11 transformation event, corn plants expressing a Cry1Fprotein, cotton plants expressing a Cry1Ac protein or cotton plantsexpressin a Cry1Ac and a Cry2Ab protein (Bollgard™ I or II), cottonplants expressing a Cry1F, Cry1F/1Ac hybrid protein or a VIP3A protein,and corn or cotton plants combining such transgenic events in the sameplant species.

Preferred embodiments of the invention are shown in the below Exampleswhich are a selection of possible embodiments and an illustration of theinvention. It is evident that a variety of noncritical parameters can bechanged to give the same or essentially the same result. All referencesand published patent applications cited in this application areincorporated herein by reference.

The following sequences are enclosed to this application in the sequencelisting:

SEQ ID NO: 1—designed degenerate primer DNA sequence of eIF1A-F primer

SEQ ID NO: 2—designed degenerate primer DNA sequence of eIF1A-R primer(n at position 20 is uncertain and can be a, c, g, or t)

SEQ ID NO: 3—designed degenerate primer DNA sequence of alpha-tubulin-Fprimer

SEQ ID NO: 4—designed degenerate primer DNA sequence of alpha-tubulin-Rprimer

SEQ ID NO: 5—DNA sequence of the A. gossypii eIF1A gene PCR fragment

SEQ ID NO: 6—DNA sequence of the M. persicae eIF1A gene PCR fragment

SEQ ID NO: 7—DNA sequence of the A. gossypii alpha-tubulin gene PCRfragment (n at positions 591, 592 and 637 is uncertain and can be a, c,g, or t)

SEQ ID NO: 8—DNA sequence of the M. persicae alpha-tubulin gene PCRfragment (n at positions 3, 113, 128, 137, 509, 615, 617, and 627 isuncertain and can be a, c, g, ort)

SEQ ID NO: 9—designed primer sequence of the Myzus persicae EcR C domainforward primer

SEQ ID NO: 10—designed primer sequence of the Myzus persicae EcR Edomain reverse primer

SEQ ID NO: 11—DNA sequence of the Myzus persicae EcR PCR fragment

SEQ ID NO:12—DNA sequence of the Myzus persicae alpha-actinin genefragment (n at position 704 is uncertain and can be a, c, g or t).

Unless stated otherwise in the Examples, all recombinant DNA techniquesare carried out according to standard protocols as described in Sambrookand Russell (2001) Molecular Cloning: A Laboratory Manual, ThirdEdition, Cold Spring Harbor Laboratory Press, NY, in Volumes 1 and 2 ofAusubel et al. (1994) Current Protocols in Molecular Biology, CurrentProtocols, USA and in Volumes I and II of Brown (1998) Molecular BiologyLabFax, Second Edition, Academic Press (UK). Standard materials andmethods for plant molecular work are described in Plant MolecularBiology Labfax (1993) by R. D. D. Croy, jointly published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications,UK. Standard materials and methods for polymerase chain reactions can befound in Dieffenbach and Dveksler (1995) PCR Primer: A LaboratoryManual, Cold Spring Harbor Laboratory Press, and in McPherson at al.(2000) PCR—Basics: From Background to Bench, First Edition, SpringerVerlag, Germany. Detailed information and references on RNA interferencecan be found on the website of Ambion, at: www.ambion.com.

EXAMPLES Example 1 Injection and Feeding of Aphids with dsRNA InsectRearing

Aphis gossypii were raised on 3-5 week old cotton plants, at 25° C.under a 14 hour:10 hour day/night cycle, with low to medium humidity.Between 5 and 10 apterae adults were placed on each plant on a weeklybasis. Myzus persicae were raised on 3-5 week old radish plants, at 20°C. under a 14 hour:10 hour day/night cycle with low to medium humidity.Between 10 and 15 apterae adults were placed on each plant every twoweeks.

PCR Amplification and Sequence Analysis of Aphid Genes

Total RNA was extracted from a population of aphids of mixed life stagesusing a Qiagen RNeasy Minikit™, according to the manufacturer'sspecifications. The RNA (−250 ng) was used to prepare cDNA usingInvitrogen's Thermoscript RT-PCR System™, using oligo(dT) primers. Theresulting cDNA was then used as template for the amplification of theeukaryotic initiation factor.

Degenerate PCR primers were designed to span moderate to highlyconserved regions of the eukaryotic initiation factor 1A (eIF1A) geneand the alpha-tubulin genes, based on alignments of multiple sequencesof invertebrate's genes derived from GenBank. Sequences were aligned andmultiple sequence comparisons were generated using either the GCGprogram ‘Pileup’ or ‘CLUSTAL W’ with default parameters for thenucleotide sequences and the default-scoring matrix for proteins.Primers were designed to cover a single putative exon whenever possible.Exon predictions were usually based on exon boundaries found in theDrosophila melanogaster orthologue of the target gene. To assist withthe design process, the CODEHOP program (Blocks Server,http://www.blocks.fhcrc.org) was used. TABLE 1 PCR primers used toisolate gene sequences from A. gossypii and M. persicae Primers Primersequence eIF 1A primers: EIF1A-F (SEQ ID NO:1) 5′ AAA ACA GAA GAA GAGGTA AAA AYG ARA 3′ EIF1A-R (SEQ ID NO:2) 5′ GGT TTC TGG CTT CGT CTG GNGTRT AYT T 3′ Alpha-tubulin primers: Tubulin-F (SEQ ID NO:3) 5′ TAC AACTCS ATC YTG ACC AC 3′ Tubulin-R (SEQ ID NO:4) 5′ TCC ATR CCY TCW CCB ACRTAC C 3′

Amplifications of the target genes were performed using Invitrogen'sThermoscript RT-PCR System, with Platinum Taq DNA polymerase, on aCorbett Research Thermocycler. A range of annealing temperatures (0.5 to5 degrees below the predicted T_(m) of the primers), and a range ofMgCl₂ concentrations (0 to 3.0 mM) were tested to find the conditionsthat produced the maximum amount of specific PCR product. The followingPCR conditions were used: 94° C. for 2 min, 30 cycles of [94° C. for 30sec; 47-52° C. for 30 sec; 72° C. for 90 sec], and 72° C. for 3 min.

The PCR products were gel purified using a Perfectprep Gel Cleanup Kit(EppendorF) and subcloned into the plasmid pGEM-T-Easy (Promega).Isolated plasmid DNA was sequenced using primers complementary to the T7and SP6 promoters in the vector. DNA was sequenced usingBigDye-terminator chemistry (Applied BioSystems) according to themanufacturer's specifications, and sequencing reactions were resolvedand processed by an Applied BioSystems Model377XL automated DNAsequencer.

DNA sequences were edited to remove plasmid sequences and pairwisecomparisons were performed using the GCG alignment program ‘Gap’(Devereux et al., 1984) and multiple sequence comparisons and consensussequences were generated using either the GCG program ‘Pileup’ (Devereuxet al., 1984) or ‘CLUSTALW’ (Thompson et al., 1994) with the defaultparameters (gap weight 5.0, gap length weight 0.3) for nucleotidesequences.

A single 279 nt long PCR product was amplified from A. gossypii and M.persicae cDNA using the aforementioned eIF primers. Each PCR product wassequenced, and based on DNA sequence comparisons with other knowninvertebrate eIF genes, both were confirmed to be eIF gene fragments.SEQ ID NO: 5 and 6 show the DNA sequences of the A. gossypii and M.persicae eIF1A PCR fragments, respectively.

The sequences of the alpha-tubulin A. gossypii and M. persicae PCRfragments corresponding to the genes found in the cDNA library of theseinsects is shown in SEQ ID NO: 7 and 8, respectively.

Using the above protocol, also DNA sequences for the A. gossypiialpha-actinin, acetylcholinesterase and elongation factor 1-alpha geneare determined, based on the available sequences of the Drosophilaorthologs of these gene in Genbank accession numbers NM058137(alpha-actinin), X05893 (acetylcholinesterase), or based on theavailable sequence of the numbers M. Persicae ortholog of the elongationfactor 1 alpha gene in Genbank accession number AF143612.

Using a similar procedure as described above for the eIF andalpha-tubulin genes, also a DNA sequence of a Myzus persicaealpha-actinin gene was isolated. This sequence is shown in SEQ ID NO:12.

Preparation of Double-Stranded RNA

To facilitate in vitro dsRNA synthesis, the gene fragment from thepGEM-T-Easy plasmid was subcloned into the pL4440 plasmid (Timmons andFire, 1998), a plasmid with two convergent T7 promoters. Transcriptionfrom this plasmid requires the use of only T7 RNA polymerase to produceboth sense and antisense RNAs simultaneously, which will anneal in vitroto form dsRNA.

The plasmid pL4417 (described in Fire Lab 1997 Vector Supplement,February 1997, provided by Andrew Fire, Carnegie Institute; Timmons etal., 2001) contains the Aequorea victoria green fluorescent proteingene, gfp, flanked by two convergent T7 promoters. This plasmid was usedto prepare dsRNA of the entire (716 nt) open reading frame of the gfpgene, to assess whether non-aphid specific dsRNA had any deleteriouseffects on the aphids.

To obtain sufficient DNA template for in vitro transcriptions, the aphidgene fragment and flanking T7 promoter sequences were PCR-amplified fromthe pL4440 plasmid using plasmid-specific primers.

Double-stranded RNA was prepared using a T7 RiboMAX Express Large ScaleRNA production System (Promega) according to the manufacturer'sspecifications.

Insect Bioassays

To test if the A. gossypii eIF1A dsRNA was functional in silencing theaphid gene, haemocoel injections of dsRNA targeting this aphid gene weredone in A. gossypii adult females and the mortality in time and thefecundity was compared to control, buffer-injected or dsGFP (greenfluorescent protein)-injected A. gossypii. 25 apterae adults wereinjected with 1 microg/microl dsRNA in injection buffer (5 mM KCl, 0.1 Msodium phosphate, pH 6.8). Ten apterae adults were similarly injectedwith dsGFP (1 microg/microl) in injection buffer and 10 apterae adultswere injected with injection buffer only. Injected aphids were placedindividually in arenas containing the artificial aphid diet describedbelow. Mortality of the adults and the number of live nymphs wererecorded at 3, 7, 11, and 14 days post injection, after which theexperiment was terminated. This regime was repeated three times suchthat a total of 75 adults had been injected with the aphid-specificdsRNA. Injection of eIF dsRNA resulted in a considerable percentage(29%) of A. gossypii adults dying within 3 days following the injection,and the remaining injected aphids continued to die over the following 2weeks. In comparison, considerable fewer aphids died at 3 days postinjection when injected with either GFP dsRNA (3%) or injection buffer(7%).

Injections of M. persicae with M. persicae eIF dsRNA also killed someaphids, but the mortality rates in this experiment did not appear to besignificant when compared to GFP dsRNA- or buffer-injected controls (alarge variation was observed in the controls and the treatment).

For those A. gossypii females that survived the injection treatment,their nymph production was assessed over a two week period. Thecumulative number of nymphs produced at 7, 11, and 14 days postinjection were similar for all treatments, with the aphid-specific dsRNAhaving no significant effect on the reproduction of the treated femalesthat survived.

Similarly, a portion of the Myzus persicae ecdysone receptor (EcR) genewas amplified from M. persicae cDNA using the following PCR primersequences: MpEcR C domain forward primer: 5′ CCCAAGCTTTGCCTGGTGTGTGGC(SEQ ID NO:9) GACCGG 3′. MpEcR E domain reverse primer: 5′CCCAAGCTTATCCTGGAAATAGAC (SEQ ID NO:10) AAGTCG 3′.

The underlined adapters were added to provide a HindIII site forsubcloning into a dsRNA expression vector.

The about 450 bp PCR product was gel-purified using a Perfectprep GelCleanup Kit (Eppendorf) and subcloned into the plasmid pGEM-T-Easy(Promega). The gene fragments from the pGEM-T-Easy plasmid weresubcloned into the pL4440 plasmid (kindly provided by Andrew Fire), aplasmid with two convergent T7 promoters. Transcription from thisplasmid requires the use of only T7 RNA polymerase to produce both senseand antisense RNAs simultaneously, which will anneal in vitro to formdsRNA. Double-stranded RNA was prepared using a T7 RiboMAX Express LargeScale RNA production System (Promega) according to the manufacturer'sspecifications.

SEQ ID NO:11 shows the Myzus persicae ecdysone receptor DNA sequencecoding from the start of the C domain to the 15th residue of the Edomain (about 450 bp).

Two to three day old adult females were microinjected into theirabdominal haemocoels with 50 nL of buffer, with or without 1 ug/uL dsRNA(eIF1A or EcR). Aphids were anaesthetised using CO2, and secured to amicroscope slide coated with a dried, sticky layer of 20% sucrose. Theinsects were injected using borosilicate glass needles, prepared using amicropipette puller (Sutter Instruments) and sometimes sharpened with aneedle beveller (Narashige). The needle was operated using amicromanipulator (Narashige) secured to a stereomicroscope, and thefluid was delivered with the aid of an air-driven pump controlled by afoot-operated solenoid switch. Injected aphids were washed from thesucrose pad, and transferred to individual feeding arenas. The arenasconsisted of small (1.5 cm inner diameter) plastic cylinders closed atone end with a glass microscope coverslip and at the other end with twoParafilm membranes. An artificial liquid diet (see below) was containedbetween the two Parafilm membranes, and the survival of the injectedaphids, and the number of nymphs that they produced over a 10-day periodwas monitored.

No increase in aphid mortality was observed for M. persicae aphidsinjected with EcR dsRNA. Aphids injected with buffer only or with eIFdsRNA produced a similar number of nymphs at days 5 and 10 postinjection (FIG. 1). Aphids injected with EcR dsRNA producedsignificantly fewer nymphs after day 10 (n=25, Student's T-test,P<0.01), suggesting that although the injected dsRNA had taken some timeto promote an effect on nymph production, it adversely affected insectfecundity.

Also, injection of Aphis gossypii alpha-tubulin dsRNA in the haemocoelof these aphids shows that aphid mortality is increased compared tocontrol dsGFP injections. Hence, the A. gossypii tubulin sequence of SEQID NO: 7 or the gene corresponding to this sequence is another possibletarget gene.

For the feeding assays, three to four adult female A. gossypii aphidswere placed in individual feeding arenas and left for 24 h. The adultswere then removed, leaving the newborn nymphs to develop entirely on theartificial diets containing dsRNA. The arenas consisted of small (1.5 cminner diameter) plastic cylinders closed at one end with a glassmicroscope coverslip or plastic film and at the other end with twoParafilm membranes. A total of 100 microliter of artificial liquid dietwas contained between the two Parafilm membranes, containing either 1microgram/microliter dsRNA (gfp or eIF) or 10 microliter RNA dilutionbuffer (10 mM Tris, pH 8.0). Three to five independent preparations ofeIF and gfp dsRNA were pooled to provide sufficient quantities of dsRNA(5 mg) for the feeding bioassays. The artificial diet was prepared as a2× stock, so that following addition of dsRNA and buffer, the finalconcentration of diet could be diluted to 1× concentration by adding theappropriate volume of water. Between 45 and 50 arenas were set up foreach treatment (buffer, gfp-dsRNA, and eIF-dsRNA), starting with, onaverage, 40 nymphs per arena at day 1. The number of live aphids at days3, 7, and 11 were recorded, and the percentage of aphids surviving foreach arena, relative to day 1, was calculated.

The artificial diet used includes, for 9 ml (all ingredients from cellculture quality): amino acid stock solution (1 ml), vitamin stocksolution (0.3 ml), sucrose 80% (3.125 ml), potassium-sodium phosphatebuffer (1 ml), ovalbumin (10 mg), MgCl₂ (10 mg), Wesson salts (10 mg),water (3.57 ml). The potassium-sodium buffer solution (100 mM, pH 7.0)contains: NaH₂PO₄: 1.4 g, K₂HPO₄: 2.6 g, Ascorbic Acid: 0.25 g, adjustpH to 7.0, mix with 250 ml water (this is not stored, an amount is madeto use immediately in the diet which is stored). The diet is mixed,filtered on 0.2 μm filters and stored in 5 ml tubes at −70° C. A largeamount is prepared, which is stored in the freezer for ±6 months. Justbefore a bioassay, antibiotics are added: to 9 ml of diet, 20 μlerythromycine (10 mg/ml EtOH), 10 μl triacilline (100 mg/ml), 5 μlchloramphenicol (60 mg/ml), 6 μl kanamycine (50 mg/ml). This has somenegative effect on growth of larvae and can be omitted when working withcell-free samples.

The amino acid stock solution contains (all L-amino acids, betweenbrackets final concentration in mM are indicated): Glutamine (32.8),Serine (25.5), Arginine (10.7), Valine (7.1), Threonine (6.9), Leucine(5.1), Lysine (5.1), Isoleucine (4.4), Phenylalanine (2.8), Histidine(2.2), Methionine (1.6), Alanine (1.6), Tryptophan (0.9).

The vitamin stock solution contains (between brackets mg/10 ml): biotin(0.1), Ca-pathotenate (5), choline chloride (50), myo-inositol (50),niacin (10), pyridoxin (2.5), thiamine-HCl (2.5).

The amino acid stock solution and the vitamin stock solution are notstored: an amount is prepared to use immediately in the diet which isstored.

Feeding eIF1A dsRNA to A. gossypii nymphs resulted in a significantreduction in the survival of nymphs (FIG. 2). After 11 days, only 17(±1.4) % of the aphids fed on diet containing eIF dsRNA had survived,whereas 54 (±4.1) and 51 (±2.8) % of the aphids fed on diet containingbuffer or gfp dsRNA had survived. Hence, there is a two-fold increase inaphid mortality due to the presence of the eIF dsRNA in the diet.Student t-tests confirmed that the feeding of eIF dsRNA significantlyreduced the survivorship of aphids, relative to aphids fed either bufferor gfp dsRNA (P<0.0001).

These data of a significantly higher mortality of aphids when fed dsRNAhave been confirmed in another experiment wherein a smaller number ofaphids was used, but using a similar experimental setup as above (alsousing the A. gossypii eIF1A dsRNA fed to A. gossypii nymphs).

A similar experimental setup with M. persicae aphids and a dsRNA basedon the above M. persicae eIF1A sequence (SEQ ID NO:6) confirms that asignificant mortality is obtained when feeding these aphids naked,unpackaged dsRNA molecules in their diet.

These results show that surprisingly, significant mortality is found inaphids fed naked, unpackaged dsRNA targeted to an essential aphid gene.

Also, insect mortality and efficient silencing is obtained by usingsmall interfering RNA molecules (siRNA) directly in the aphid diet. For3 target genes (eIF, tubulin and GFP), siRNA is prepared by long dsRNAcleavage using the RNAseIII (Ambion's Silencer siRNA Cocktail Kit,generating 12-30 bp siRNAs) or recombinant human dicer (Gene TherapySystems, producing (more optimal) 21-23 bp siRNAs). Aphids are fedartificial diet containing a concentration of long dsRNA and siRNAs todetermine the difference in efficacy. Application of siRNA molecules inthe aphid diet also produces a significant effect on aphid development,comparable to or even better then the longer dsRNA applied above.

Addition of a poly-Arginine 12-mer cationic oligopeptide to the aphiddiet, together with the eIF1A dsRNA, results in a significantlyincreased mortality in the above feeding assays towards A. gossypii andM. persicae, compared to the application of dsRNA without such peptideand a control setup using GFP dsRNA.

Example 2 Analysis of Aphid Feeding on Plants Transiently ExpressingdsRNA

To confirm that plants can deliver dsRNA or siRNA to plant sap-suckinginsects such as aphids in planta without transfection promoting agents,aphids are added to tobacco plants transiently induced to produce aphiddsRNA using the SVISS methodology described in published PCT applicationWO 03/052108 (incorporated herein by reference), using dsRNA targeted toa M. persicae essential gene.

These experiments confirm that the dsRNA transiently produced in plantsin an unpackaged form, free from transfection-promoting agents, canresult in a significant inhibition of M. persicae development.

Example 3 Analysis of Aphid Feeding on Plants Stably Expressing dsRNA inPhloem

In this experiment, Arabidopsis and tobacco plants are transformed byAgrobacterium-mediated transformation with a vector containing a dsRNAchimeric gene targeting either the M. persicae eIF1A gene, the M.persicae ecdyson receptor (EcR) or the control GFP gene. A M. persicaeecdyson receptor DNA sequence was cloned from this aphid as describedabove. A plant dsRNA chimeric gene is made wherein either thephloem-specific rolC promoter (Pandolfini et al., 2003) or theconstitutive CaMV 35S promoter is operably linked to the dsRNAconstruct, containing sense and antisense regions to the above targetaphid gene or the control gene. Plants are tested for transformationusing Southern blot analysis and for expression and dicing of the dsRNAby small RNA Northern blot assay. Transport of interfering RNAs to thephloem is confirmed by the significant reduction in development of Myzuspersicae feeding on the successfully transformed Arabidopsis and tobaccoplants. Northern blot analysis confirms the presence of the RNAmolecules of the invention in the phloem sap of plants that aresuccessfully transformed.

Hence, plant sap-sucking insects can now be controlled using specificand selective dsRNA-sequences targeting essential plant sap-suckinggenes so as to minimize population build-up of aphids on crop plants.

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1. A chimeric gene comprising the following operably linked DNA: (a) aplant-expressible promoter; (b) a DNA region which when transcribedyields a double-stranded RNA molecule capable of reducing the expressionof an essential gene of a plant sap-sucking insect, said RNA moleculecomprising a first and second RNA region wherein: (i) said first RNAregion comprises a nucleotide sequence of at least 19 consecutivenucleotides having at least about 94% sequence identity to thenucleotide sequence of said endogenous gene; (ii) said second RNA regioncomprises a nucleotide sequence complementary to said at least 19consecutive nucleotides of said first RNA region; (iii) said first andsecond RNA region are capable of base-pairing to form a double strandedRNA molecule between at least said 19 consecutive nucleotides of saidfirst and second region; (c) optionally, a 3′ end region comprisingtranscription termination and polyadenylation signals functioning incells of said plant.
 2. The chimeric gene of claim 1, wherein saidessential of said plant sap-sucking insect is selected from the groupconsisting of genes encoding the following: a gut cell protein, amembrane protein, an ecdyson receptor, a γATPase, an amino acidtransporter, a transcription factor, a peptidylglycine alpha-amidatingmonooxygenase; a cystein protease, an aminopeptidase, a dipeptidase, asucrase/transglucosidase, a translation elongation factor, an eucaryotictranslation initiation factor 1A, a splicing factor, an apoptosisinhibitor; a tubulin protein, an actin protein, an alpha-actininprotein, a histone, a histone deacetylase, a cell cycle regulatoryprotein, a cellular respiratory protein; a receptor for aninsect-specific hormonal signal, a juvenile hormone receptor, an insectpeptidic hormone receptor; a protein regulating ion balance in a cell, aproton-pump, a Na/K pump, an intestinal protease; an enzyme involved insucrose metabolism, a digestive enzyme, a trypsin-like protease and acathepsin B-like protease.
 3. The chimeric gene of claim 1, wherein saiddouble-stranded RNA silences the gene corresponding to the DNA sequenceof any one of SEQ ID NO: 5 to 8, SEQ ID NO: 11 or SEQ ID NO:
 12. 4. Thechimeric gene of claim 1, wherein between said first and second RNAregion, a spacer region containing a plant intron is present.
 5. Thechimeric gene of claim 1, wherein said essential gene has a portionwhich occurs with the same sequence or with at least 94% sequenceidentity in homologous genes of several plant sap-sucking insects. 6.The chimeric gene of claim 1, wherein said promoter is a constitutivepromoter.
 7. The chimeric gene of claim 1, wherein said promoter is avascular-specific or a phloem-specific promoter.
 8. The chimeric gene ofclaim 7, wherein vascular- or phloem-specific promoter is selected fromthe group consisting of: a rolC or rolA promoter of Agrobacteriumrhizogenes, a promoter of a Agrobacterium tumefaciens T-DNA gene 5, therice sucrose synthase RSs1 gene promoter, a Commelina yellow mottlebadnavirus promoter, a coconut foliar decay virus promoter, a ricetungro bacilliform virus promoter, the promoter of a pea glutaminesynthase GS3A gene, a invCD111 and invCD141 promoters of a potatoinvertase genes, a promoter isolated from Arabidopsis shown to havephloem-specific expression in tobacco by Kertbundit et al (1991), aVAHOX1 promoter region, a pea cell wall invertase gene promoter, an acidinvertase gene promoter from carrot, a promoter of a sulfate transportergene Sultr1;3, a promoter of a plant sucrose synthase gene, a promoterof a plant sucrose transporter gene.
 9. A plant cell, tissue, or a plantor a plant seed comprising the chimeric gene or the double-stranded RNAmolecule described in claim
 1. 10. A method to silence a gene of a plantsap-sucking insect, comprising applying to the feed of said plantsap-sucking insect unpackaged, naked dsRNA or siRNA which is targeted toan essential plant sap-sucking gene.
 11. The method of claim 10, whereinsaid essential gene is any of the genes listed in claim 2 above.
 12. Themethod of claim 10, wherein said application is by expression of a dsRNAchimeric gene in phloem cells of a plant.
 13. A method to silence a genein an plant sap-sucking insect, comprising: adding naked, unpackageddsRNA or siRNA to the diet or feed of said plant sap-sucking insect,wherein said dsRNA or siRNA targets said gene.
 14. A method ofcontrolling plant sap-sucking insects, comprising expressing in thephloem of a plant dsRNA that targets an essential plant sap-suckinginsect gene.
 15. The method of claim 14 wherein said gene is a geneexpressed at least in the intestine or in gut cells.
 16. The method ofclaim 14 wherein said plant sap-sucking insect is an aphid or awhitefly.
 17. A plant, comprising stably inserted in its genome, thechimeric gene of claim 1, so that said chimeric gene is expressed in thephloem or xylem of said plant.
 18. A method of identifying gene functionin a plant sap-sucking insect, comprising the step of applying naked,unpackaged dsRNA targeting a plant sap-sucking insect gene to the dietof said insect, and evaluating phenotypic or biochemical changes in saidinsect.
 19. A method of identification of novel targets for insecticidalcompounds, comprising the steps of: a) applying naked, unpackaged dsRNAor siRNA molecules to the feed or diet of a plant-sap sucking insect; b)analyzing which genes when silenced confer lethality to said insect, c)cloning and characterizing said genes thus analyzed; d) identifyingcompounds that disrupt or inactivate said gene or the RNA or proteinencoded thereby; and e) contacting said compounds with said insect orfeed or diet of said insect to confirm the pesticidal nature of saidcompound.
 20. Phloem of a plant, comprising siRNA targeted to an aphidessential gene.
 21. Phloem sap of a plant, comprising siRNA targeted toan aphid essential gene.
 22. An aphid gene comprising the sequence ofany one of SEQ ID NO:5 to 8, SEQ ID NO: 11 or SEQ ID NO:
 12. 23. Themethod of claim 18, wherein a cationic oligopeptide is mixed in the diettogether with the dsRNA.
 24. The method of claim 23, wherein saidoligopeptide is a 12 amino acids poly-Arginine peptide.
 25. The plantcell, tissue, plant or plant seed of claim 9, which also comprises achimeric gene encoding a cationic oligopeptide.
 26. The plant cell,tissue, plant or plant seed of claim 25, wherein said oligopeptide is a12 amino acids poly-Arginine peptide.
 27. The method of claim 19,wherein a cationic oligopeptide is mixed in the diet together with thedsRNA.
 28. The plant cell, tissue, plant or plant seed of claim 17,which also comprises a chimeric gene encoding a cationic oligopeptide.